Compositions and methods for the treatme of primary and metastatic neoplastic disea using arsenic compounds

ABSTRACT

The invention relates to the use of arsenic compounds to treat a variety of neoplastic diseases. The present invention encompasses the administration to a mammal of arsenic in the form of a salt, complex, organic compound or ionic solution to treat tumors of epithelial tissue, connective tissue, central nervous system, lymphoid tissue, hematopoietic cells and tumors associated with oncogenic viruses. This invention also encompasses the treatment of hematopoietic disorders in mammals by the administration of one or more arsenic compounds to said mammal. Further, the arsenic compounds may be used to treat metastatic neoplastic diseases.

1. FIELD OF INVENTION

[0001] The present invention relates to methods and compositions for thetreatment of primary and metastatic neoplastic diseases, including, butnot limited to human sarcomas, carcinomas and hematopoietic disorders.In the practice of the treatment of cancer, compositions containingarsenic compounds are used to arrest and reverse neoplastic growth.

[0002] More specifically, the present invention relates to novelchemotherapeutic methods—novel uses of arsenic compounds for treatingprimary and metastatic tumors; primary and metastatic tumors of thecentral nervous system; refractory primary and metastatic tumors of thecentral nervous system; breast, lung, bladder and prostate cancer; andrefractory breast, lung, bladder and prostate cancer to mention a few.

2. BACKGROUND OF THE INVENTION

[0003] In 1997 more than one million people will develop some type ofcancer in the United States. Approximately 500,000 will be cured or in astate of remission. These numbers represent an improving cure rate seenover the past decade which is largely due to earlier detection, bettertreatment and advances in chemotherapy. In particular, the advances inchemotherapy include targeted or specific drug therapy in which a drugis developed specifically for the treatment of a certain cancer type.This “disease-oriented” approach is designed to identify compounds whichexert selective effects in vitro on particular tumor types and tofollow-up these leads in vivo utilizing cell lines, (Fiebig et al.,Cancer Treatment Reviews 17:109-117 (1990)). However, the incidence ofcancer is continuing to climb as our population ages and as new cancersdevelop or occur more frequently, such as in patients infected with AIDSvirus. Thus, it is clear that there is a tremendous demand foradditional regimens to treat patients with cancer.

2.1. Pathobiology of Cancer

[0004] Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

[0005] Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

[0006] The neoplastic lesion may evolve clonally and develop anincreasing capacity for invasion, growth, metastasis, and heterogeneity,especially under conditions in which the neoplastic cells escape thehost's immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993,Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).

2.2. AIDS-Related Non-Hodgkin's Lymphoma

[0007] Since the discovery of AIDS, the disease has had a closeassociation with an interesting spectrum of cancers. Further, the typesof malignancies and their incidence rates are increasing as thedevelopment of effective antiretroviral therapies and prophylaxisagainst opportunistic infections leads to prolonged survival in theimmunodeficient state for AIDS patients, (Karp and Broder, Cancer Res.51:4747-4756 (1991)). AIDS-related non-Hodgkin's lymphoma was found tooccur in AIDS patients only after 1981. AIDS-related non-Hodgkin'slymphoma is a very aggressive disease with a very high incidence ofcentral nervous system involvement. It is increasing in incidence in theAIDS population. As patients infected with the AIDS virus now livelonger because they are not dying of the usual infections, they aredeveloping lymphoma at an increasing rate. The characteristics ofAIDS-related non-Hodgkin's lymphoma are detailed in an article by Karpand Broder, (1991), supra.

[0008] The problems the medical oncologist has in treating patients withAIDS-related lymphomas is the recently described predilection foroccurrence of the lymphoma in the central nervous system (in brain andsurrounding meninges) and the fact that the patient with AIDS has a veryweak bone marrow which cannot tolerate treatment with standardchemotherapy. This makes the treatment of lymphoma in patients with AIDSvery difficult because standard chemotherapeutic agents are usually verymarrow suppressive and do not cross blood brain barrier (to treat thecentral nervous system disease).

2.3. Primary and Metastatic CNS Tumors

[0009] The incidence of primary and metastatic brain tumors isincreasing in the United States. Indeed, the arsenal ofchemotherapeutics for these types of cancers is minimal, while the needfor such therapeutics is high.

[0010] Glioblastoma multiform and other primary and metastatic centralnervous system tumors are devastating malignancies. The treatment ofthese tumors include surgery, radiation therapy and treatment withagents such as the nitrosourea BCNU. Other chemotherapeutic agentsutilized include procarbazine, vincristine, hydroxyurea and cisplatin.Unfortunately, even when all three modalities (surgery, radiationtherapy and chemotherapy) are utilized, the average survival of patientswith central nervous system malignancies is still about 57 weeks.Clearly, new treatment approaches are needed both for patients withnewly diagnosed primary and metastatic central nervous system tumors, aswell as for patients with such tumors which are refractory to the abovemodalities. Finding such new agents has been complicated by the factthat there is no animal model which appears to predict what agent willbe clinically effective against primary and metastatic central nervoussystem tumors.

2.4. Breast, Lung, Bladder and Prostate Cancers

[0011] Breast cancer has been known to occur in about one in every 8-9women in the United States. The treatment for early breast cancer issurgery, with or without radiation therapy, or surgery, with or withoutradiation therapy, plus chemotherapy and/or hormonal therapy. Despitethe best efforts of physicians there are still more than 80,000 deathseach year from breast cancer and the incidence is still rising. Currentchemotherapy for patients with primary or metastatic breast cancerincludes treatment with cyclophosphamide, methotrexate, doxorubicin,5-fluorouracil, cisplatin, vinblastine, taxol, taxotere, mitomycin C andoccasionally other agents. Unfortunately, even with these agents, almostall women who develop metastatic breast cancer succumb to their disease.One particular place that metastatic breast cancer does metastasize tois the central nervous system. When central nervous system metastases dooccur, the usual treatment is surgery (for a solitary metastasis) orradiation, or surgery plus radiation therapy. At present there is nochemotherapy which is felt to be helpful in this situation.

[0012] Lung cancer is responsible for more than 150,000 deaths each yearin the United States. Most patients with lung cancer present a tumorthat has already metastasized to a variety of organs, including lung,liver, adrenal gland and other organs. The current treatment formetastatic lung cancer is not yet standardized (Ihde, Daniel C.,“Chemotherapy of Lung Cancer”, The New England Journal of Medicine327:1434-1441, 1992 November 12th issue). However, chemotherapy regimenswhich are utilized include treatment with cisplatin plus etoposide,combinations of cyclophosphamide plus doxorubicin plus cisplatin, andsingle agents alone or in combination, including ifosfamide, teniposide,vindesine, carboplatin, vincristine, taxol, nitrogen mustard,methotrexate, hexamethylmelamine and others. Despite thesechemotherapeutic regimens the average patient with metastatic lungcancer still only survives 7-12 months. One particular troublesome placefor metastases of lung cancer is the central nervous system. Thetreatment for central nervous system metastases includes surgery (toremove a solitary lesion), radiation therapy, or a combination of both.Unfortunately, there is not standard chemotherapy which is felt to behelpful in this situation.

[0013] Each year about 11,000 patients die of bladder cancer in the U.S.Although at presentation the disease is usually localized, most patientsdevelop distant metastatic disease. The most recent advances have beenin the area of chemotherapy for patients with such metastatic disease.One effective regimen is called the MVAC regimen. It consists oftreatment with methotrexate plus vinblastine plus adriamycin(doxorubicin) plus cisplatin. Although the response rate is high to thischemotherapeutic regimen, medical oncologists are noting that one placethe patients fail is with metastases to the central nervous system.Unfortunately, there is no standard chemotherapy which is felt to behelpful in this situation.

[0014] It is estimated that more than 100,000 men will be diagnosed withprostate cancer this year and more than 30,000 patients will die fromthe disease. The most common sites of metastases in patients withprostate cancer are the bone and lymph nodes. The bone metastases areparticularly bothersome in that they can create intense pain for thepatient. The current treatment for metastatic prostate cancer includestreatment with flutamide, leuprolide, diethylstilbestrol, and otherhormonal manipulations, as well as chemotherapy (doxorubicin,estramustine phosphate, vinblastine, suramin, cisplatin, and others).Unfortunately, none of these agents are consistently helpful in thedisease. In addition, as patients with prostate cancer live longer withtheir malignancy, they will most likely develop a higher incidence ofmetastases to the central nervous system (including the spinal cord).

[0015] In general, as patients are living longer with the commonmalignancies such as breast cancer, lung cancer, bladder cancer,prostate cancer and a variety of other malignancies (because of controlof their systemic disease with surgery, radiation therapy andchemotherapy), oncologists are noting that they are developing anincreasing incidence of metastatic tumors in the central nervous systemincluding the brain. This is probably because most of the currentlyavailable chemotherapy does not cross the blood brain barrier. When thepatient (who has their tumor controlled in sites outside of the brain)develops brain metastases, it is a very difficult situation. Options forthat patient are usually limited to surgery for a solitary metastasisand/or radiation therapy. However, after those modalities fail, thepatient usually has no other options.

[0016] For each of the above indications (primary brain tumors andmetastases to the brain from other common tumors such as breast, lung,bladder and prostate cancers), there is a tremendous need for a moreeffective treatment and/or methods for improving the quality of patientlife.

2.5. Esophageal Cancer

[0017] In the U.S., carcinoma of the esophagus represents about 6% ofall cancers of the gastrointestinal tract but causes a disproportionatenumber of cancer deaths. (Boring, C. C., et al.: Cancer statistics,1993. CA Cancer J. Clin. 43:7, 1993). These cancers usually arise fromthe epithelial layer of the esophagus and are either squamous cellcarcinomas or adenocarcinomas. Overall the 5 year survival is about 5%.

[0018] Squamous cell carcinoma generally occurs after the age of 50 andis more common in males than in females. The incidence varies widelyfrom country to country and between regions within countries. In theU.S. the incidence is between 2 and 8 persons per 100,000 and is moreprevalent in blacks than in whites.

[0019] Adenocarcinoma represents 25% of all esophageal CA in the U.S. Itis usually located in the distal one third of the esophagus and mayinvade the adjacent gastric cardia. It tends to occur in people over 40years of age and is more common in males than in females. It is morecommon in whites than in blacks.

2.6. Arsenic and its Medical Uses

[0020] Arsenic has been considered to be both a poison and a drug for along time in both Western and Chinese medical practices. In the latterpart of the nineteenth century, arsenic was used frequently in attemptsto treat diseases of the blood in the West. In 1878, it was reportedthat treatment of a leukemic patient with Fowler's solution (a solutionof potassium arsenite) reduced markedly the count of white blood cells(Cutler and Bradford, Am. J. Med. Sci., January 1878, 81-84). Furtherinterests in the use of Fowler's solution as a palliative agent to treatchronic myelogenous leukemia (CML) was described by Forkner and Scott in1931 (J. Am. Med. Assoc., 1931, iii, 97), and later confirmed byStephens and Lawrence in 1936 (Ann. Intern. Med. 9, 1488-1502).Typically, Fowler's solution was orally administered to leukemicpatients as a solution until the level of white blood cells wasdepressed to an acceptable level or until toxicities (such as skinkeratoses and hyperpigmentation) developed, while the patients enjoyedvarying periods of remission. In the 1960's, Fowler's solution was stillused occasionally in attempts to treat CML, however, most patients withCML were treated with other chemotherapeutic agents, such as busulfan,and/or radiation therapy (Monfardini et al., Cancer, 1973, 31:492-501).

[0021] Paradoxically, one of the long recognized effects of exposure toarsenic, whether the source is environmental or medicinal, is skincancer (Hutchinson, 1888, Trans. Path. Soc. Lond., 39:352; Neubauer,1947, Br. J. Cancer, 1:192). There were even epidemiological data tosuggest that the use of Fowler's solution over long periods could leadto an increased incidence of cancer at internal sites (Cuzick et al.,Br. J. Cancer, 1982, 45:904-911; Kaspar et al., J. Am. Med. Assoc.,1984, 252:3407-3408). The carcinogenicity of arsenic has since beendemonstrated by the fact that it can induce chromosomal aberration, geneamplification, sister chromatid exchanges and cellular transformation(See e.g., Lee et al., 1988, Science, 241:79-81; and Germolec et al.,Toxicol. Applied Pharmacol., 1996, 141:308-318). Because of the knowncarcinogenic effect of arsenic, its only therapeutic use in human inWestern medicine today is in the treatment of tropical diseases, such asAfrican trypanosomiasis, (melarsoprol, or Arsobal® by Rhone PoulencRorer, Collegeville, Pa.; See Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th edition, chapter 66, 1659-1662, 1997).

[0022] In traditional chinese medicine, arsenous acid or arsenictrioxide paste has been used to treat tooth marrow diseases, psoriasis,syphilis and rheumatosis (Chen et al., 1995, in Manual of ClinicalDrugs, Shanghai, China, Shanghai Institute of Science and Technology,p.830). In 1970s, arsenic trioxide had been applied experimentally totreat acute promyelocytic leukemia (APL) in China (commented by Mervis,1996, Science, 273:578). The clinical efficacy of arsenic trioxide hasrecently been re-investigated in 14 of 15 patients with refractory APL,where the use of an intravenous dose at 10 mg/day for 4-9 weeks wasreported to result in complete morphologic remission without associatedbone marrow suppression (Shen et al., 1997, Blood, 89:33543360). It wasalso reported that arsenic trioxide induced apoptosis (programmed celldeath) in vitro in NB4 cells, an APL cell line, and that apoptosis wasapparently associated with down-regulation of the oncogene bcl-2, andintracellular redistribution of the chimeric PML/RARα protein that areunique to APL cells (Chen et al., 1996, Blood, 88:1052-1061; Andre etal., 1996, Exp. Cell Res. 229:253-260). Similarly, melarsoprol has beenreported to induce apoptosis in cell lines representative of chronicB-cell leukemia (Konig et al., 1997, Blood 90:562-570). Whetherapoptosis is induced in APL patients is presently unclear, but it isbelieved by some to be one of the possible mechanisms of the therapeuticeffects of certain arsenic compounds.

[0023] Although arsenic is well known to be both a poison and acarcinogenic agent, there have been many reports concerning the use ofarsenic in medical treatment. Identification or discussion of the artabove must not be construed as an admission that such is prior art.

[0024] Further, from the above discussion, it should be clear that thereare a plethora of different types of cancers, each of which requires aunique treatment protocol. Thus, the development of a broad spectrumanti-cancer agent is extremely desirable. At a minimum, additionaleffective anti-cancer agents are needed to be added to the arsenalagainst cancer.

3. SUMMARY OF THE INVENTION

[0025] Notwithstanding the conflicting reports in the art concerningbenefits and risks of the administration of arsenic to patients,applicants have discovered that arsenic compound has broad applicabilityin the treatment of various cancers, including solid tumors and blooddisorders. For example, the present invention encompasses the use ofarsenic in the form of a salt, complex, organic compound or ionicsolution to treat tumors of epithelial tissue, connective tissue,central nervous system, lymphoid tissue, hematopoietic cells and tumorsassociated with oncogenic viruses.

[0026] Further, the present invention encompasses the use of arseniccompounds to treat mammals suffering from primary and metastaticneoplastic disease as well as infectious diseases related thereto.

[0027] In addition, this invention also encompasses the use of arseniccompounds to treat primary and metastatic breast, lung, bladder andprostate cancers in humans.

[0028] This invention also encompasses the treatment of hematopoieticdisorders in mammals by the administration of one or more arseniccompounds to said mammal. The hematopoietic disorders to be treatedinclude but are not limited to polycythemia vera, Hodgkin's Disease,non-Hodgkin's Disease including Follicular Lymphoma, Diffuse Lymphoma,lymphoblastic lymphoma, small lymphocytic lymphoma, acute lymphocyticleukemia, hairy cell leukemia, myeloid metaplasia, myeloid dysplasticsyndrome, multiple myeloma and plasmacytoma.

[0029] In accordance with the present invention, arsenic compounds canbe used alone or in combination with other known therapeutic agents(including chemotherapeutics, radioprotectants and radiotherapeutics) ortechniques to either improve the quality of life of the patient, or totreat the primary neoplastic disease. For example, the arsenic compoundscan be used before, during or after the administration of one or moreknown antitumor agents including but not limited to mustard compounds,nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil,floxuridine, methotrexate, vincristine, vinblastine, taxol, etoposide,temiposide, dactinomycin, daunorubicin, doxorubicin, bleomycin,mitomycin, cisplatin, carboplatin, estramustine phosphate, hydroxyurea,BCNU, procarbazine, VM-26 (vumon), interferons and all-trans retinoicacid (ATRA), (See for example, the Physician Desk References 1997). Inaddition, the arsenic compounds can be used before, during or afterirradiation treatment. For the treatment of HIV-infected individuals,the arsenic compounds can be used alone or in combination with AZT, ddI,ddA, ddC, d4T, 3TC and other known antiviral agents.

[0030] The invention described herein encompasses a method of treatingprimary and metastatic neoplastic diseases, a method of treating solidtumors, a method of treating leukemias, a method of treating cancersrelated to bcl-2 (oncogene), each of which comprises the administrationof a therapeutically effective and non-lethal amount of one or morearsenic compounds to a mammal in need of such therapy. The invention, asmentioned above also encompasses the use of combination therapy to treatthe aforementioned diseases.

[0031] In a particular embodiment, the arsenic compounds are used withina method to treat breast, lung, colon, ovarian, renal, non-small celllung, central nervous system, bladder, prostate and head and neck cancerby administering an effective amount of one or more arsenic compoundsalone or in combination with other antineoplastic agents or therapeutictechniques including radiotherapy and surgery.

[0032] Without being limited by any theory, the inventors believe thatthe arsenic compounds of the invention may have one or more mechanismsof action in connection with the methods described herein. For example,the arsenic compounds may act as a phosphorous analogue which interfereswith the phosphorylation events that occur in signal transductioninvolved in apoptosis. Arsenic may also act as an inhibitor ofangiogenesis, i.e., the formation of new blood vessels, thereby limitingblood flow to proliferating preneoplastic cell masses, tumors andmetastases. It is well known that if a tumor is not invaded by bloodcapillaries, it would have to depend on the diffusion of nutrients fromits surroundings and cannot enlarge beyond a certain size. Arsenic mayalso function as a differentiating agent which causes dividingpreneoplastic and/or cancer cells that display an undifferentiated orunderdifferentiated phenotype to develop into terminally differentiatedcells, and die after a finite number of cell divisions. Finally, arsenicmay also act to sensitize the cancer cells to radiation and/orchemotherapy. Thus, the arsenic compounds of the invention are describedas being useful against a variety of cancers.

[0033] Specific therapeutic regimens, pharmaceutical compositions, andkits are also provided by the invention. Thus, the invention alsoencompasses pharmaceutical compositions which comprise one or morearsenic compounds and a pharmaceutically acceptable carrier. Thecompositions are sterile solutions suitable for intravenous injection orinfusion. In another embodiment the invention encompasses a compositionsuitable for oral delivery; comprising one or more arsenic compounds anda pharmaceutically acceptable excipient or carrier. In anotherembodiment, the invention also includes compositions suitable fortopical or dermal delivery.

[0034] Particular compositions of the invention and their properties aredescribed in the sections and subsections which follow.

4. BRIEF DESCRIPTION OF THE FIGURES

[0035] FIGS. 1A-1I. Dose response curves showing percentage growth ofvarious cancer cell lines after continuous exposure to 10⁻⁵ to 10⁻⁹μg/ml of arsenic trioxide for 2 days. FIG. 1A. Leukemic cell linesCCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR. FIG. 1B. Non-SmallCell Lung Cancer cell lines A549/ATCC, EKVX, HOP-62, HOP-92, NC1-H226,NC1-H23, NCI-8322M, NC1-H460, NC1-H522. FIG. 1C. Colon Cancer cell linesCOLO 205, HCT-116, HCT-15, HT29, KM12, SW620. FIG. 1D. CNS Cancer celllines SF-268, SF295, SF-539, SNB-19, SNB-75, U251. FIG. 1E. Melanomacell lines LOX 1MV1, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5,UACC-257, UACC-62. FIG. 1F. Ovarian Cancer cell lines IGROV1, OVCAR-3,OVCAR-5, OVCAR-8, SK-OV-3. FIG. 1G. Renal Cancer cell lines A498,CAKI-1, RXE 393, SN12C, TX-10, UO-31. FIG. 1H. Prostate Cancer celllines PC-3, DU-145. FIG. 1I. Breast Cancer cell lines MCF7, NCI/ADR-RES,MDA-MB-435, MDA-N, BT-549, T-47D.

[0036]FIG. 2. Mean graphs showing selectivity patterns at each of theprincipal response parameters for all the cell lines tested aftercontinous exposure to 10⁻⁵ to 10⁻⁹ μg/ml of arsenic trioxide for 2 days.

[0037] FIGS. 3A-3I. Dose response curves showing percentage growth ofvarious cancer cell lines after continuous exposure to 10⁻⁵ to 10⁻⁹μg/ml cf arsenic trioxide for 6 days. FIG. 3A. Leukemic cell linesCCRF-CEM, K-562, MOLT-4, RPMI-8226. FIG. 3B. Non-small Cell Lung Cancercell lines EKVX, HOP-62, HOP-92, NC1-H226, NC1-H23, NC1-H322M, NC1-H460,NC1-H522. FIG. 3C. Colon Cancer cell lines COLO 205, HCT-116, HCT15,HT29, KM12, SW-620. FIG. 3D. CNS Cancer cell lines SF-268, SF-295,SF-539, SNB-75, U251. FIG. 3E. Melanoma cell lines LOX IMVI, MALMI-3M,SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62. FIG. 3F. OvarianCancer cell lines IGROVI, OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. FIG. 3G.Renal Cancer cell lines 786-0, A498, CAKI-1, RXF 393, S12C, TK-10. FIG.3H. Prostate Cancer cell lines DU-145. FIG. 3I. Breast Cancer cell linesMCF7, NCI/ADR-RIS, MDAMB-231/ATCC, HS 578T, MDA-MB-435, MDA-N, BR-549,T-47D.

[0038]FIG. 4. Mean graphs showing selectivity patterns at each of theprincipal response parameters for all the cell lines tested aftercontinous exposure to 10⁻⁵ to 10⁻⁹ g/ml of arsenic trioxide for 6 days.

5. DETAILED DESCRIPTION OF THE INVENTION

[0039] Methods and compositions for the treatment of primary andmetastatic neoplastic diseases are described herein. The invention isbased, in part, on a dosage regimen for administration of compositionscomprising arsenic. The invention is also based in part, on the potencyof the arsenic compounds of the invention against certain cancers.

[0040] This invention includes a method of treating primary solid tumorsin a mammal which comprises administering to a mammal in need of suchtherapy a therapeutically effective and non-lethal amount of one or morearsenic compound.

[0041] The invention also includes a method of treating metastatictumors in a mammal which comprises administering to a mammal atherapeutically effective and non-lethal dose of one or more arseniccompound.

[0042] The invention includes a method for treating disorders of bloodin mammal which comprises administering one or more arsenic compound ina therapeutically effective and non-lethal amount.

[0043] The arsenic compound of the invention may be utilized in in avariety of known forms; for example, arsenic can be administered as asalt, an organic or inorganic complex, an organic chelate, an organiccompound or an organic or inorganic solution. It is preferred that theform be chosen to reduce toxicity and improve efficacy. Further, theform chosen may also depend on the type and location of the tumor inquestion. The inorganic salt forms of arsenic are preferred. Forexample, inorganic salts such as arsenic triiodide, arsenic(III)bromide,arsenic(III)chloride, arsenic pentoxide, arsenic trioxide, Fowler'ssolution (potassium arsenite), sodium arsenite, and calcium arsenite maybe used. Arsenic trioxide is most preferred. Both arsenous acids andarsenites as well as arsenic acids and arsenates may be used within thepresent methods. Aqueous solutions containing arsenite ions arepreferred. Further, arsenic sulfides may be used such as arsenoussulfide, arsenic sulfide, arsenic pentasulfide, tetraarsenic trisulfideand tetraarsenic pentasulfide. Without being limited by any theory,certain of these arsenic compounds may be prodrugs to an active species.

[0044] Generally, the skilled artisan will recognize that the form ofarsenic to be used should be therapeutically effective withoutunreasonable toxicity. The toxicity is dependent upon the dose, thedosage form, the mode of administration and frequency of dosing.Generally, the skilled artisan can chose from the following known formsof arsenic: arsenic halides, arsenic oxides, arsenic acids, arsenicsulfides, and the like.

[0045] Arsenic can also be readily combined with carbon to form a widevariety of organic compounds. These include but are not limited toprimary and secondary arsines, tertiary arsines, halo arsines, dihaloarsines, cyclic and polymeric substances containing arsenic; specificexamples of organic arsenic compounds include but are not limited to3-Nitro-4-hydroxyphenylarsonic acid, arsanilic acid, sodium hydrogen4-aminophenylarsenate, melarsoprol, melarsonyl potassium, carbarsone,arsenamide arsphenamine and sodium arsanilate.

[0046] As used herein, “arsenic compound” refers to a pharmaceuticallyacceptable form of arsenic including salts, solutions, complexes,chelates and organic and inorganic compounds incorporating arsenic. Itshould be recognized that the invention includes arsenic prodrugs orcompounds that are converted in vivo to biologically active forms ofarsenic. Such prodrugs may be used to reduce or avoid the well knownpotential for arsenic toxicity. The arsenic compounds of the presentinvention can be synthesized or commercially purchased. For example, thecompounds can be prepared from well-known chemical techniques. (See forexample, Kirk-Othmer, Encyclopedia of Chemical Technology 4 ed. volume 3pps. 633-655 John Wiley & Sons).

[0047] In one embodiment, the arsenic compound of the invention isarsenic trioxide which is dissolved in an aqueous solution of sodiumhydroxide, with the pH adjusted to a physiologically acceptable range,e.g. about pH 6-8.

[0048] Any suitable mode of administration may be used in accordancewith the present invention including but not limited to parenteraladministration such as intravenous, subcutaneous, intramuscular andintrathecal administration; oral, intranasal, rectal or vaginaladministration may also be used; directly into the tumor; transdermalpatches; implant devices (particularly for slow release); finally,topical administration may be used. The mode of administration will varyaccording to the type of arsenic compound being used and the disease tobe treated.

[0049] The pharmaceutical compositions to be used may be in the form ofsterile physiologically acceptable (aqueous or organic) solutions,colloidal suspensions, creams, ointments, pastes, capsules, caplets,tablets and cachets. The pharmaceutical compositions comprising arseniccompounds of the invention can be contained in sealed sterile glasscontainers and/or ampoules. Further, the active ingredient may bemicro-encapsulated, encapsulated in a liposome, noisome or lipofoamalone or in conjunction with targeting antibodies. It should berecognized that delayed slow or sustained release forms ofadministration are also included.

[0050] The arsenic compounds of the present invention may be usedagainst a variety of primary and metastatic neoplastic diseasesincluding but not limited to primary and metastatic tumors of thecentral nervous system, breast, colon, ovaries, kidneys, lung, bladder,prostate and head and neck.

[0051] More specifically, the arsenic compounds of the present inventioncan be used to treat tumors of epithelial origin including but notlimited to:

[0052] squamous cell carcinoma

[0053] basal cell carcinoma

[0054] melanoma

[0055] tumors of epithelial lining of glands or ducts:

[0056] adenocarcinoma

[0057] papillary carcinoma

[0058] papillary adenocarcinoma

[0059] tumors of the liver and biliary tract:

[0060] Hepatocellular carcinoma

[0061] tumors of the gastrointestinal tract:

[0062] squamous cell carcinoma of the esophagus

[0063] adenocarcinoma of the esophagus

[0064] colorectal carcinoma (colon cancer)

[0065] gastric carcinoma (stomach cancer)

[0066] tumors of respiratory tract:

[0067] bronchogenic carcinoma

[0068] small cell carcinoma

[0069] large cell carcinoma

[0070] tumors of the urogenital tract:

[0071] transitional cell carcinomas of bladder

[0072] squamous cell carcinoma of bladder

[0073] carcinoma of prostate

[0074] carcinoma of cervix

[0075] tumors of breast

[0076] tumors of blood cells and related cells (leukemias):

[0077] acute and chronic lymphocytic leukemia

[0078] polycythemia vera

[0079] Cancers of Lymphoid tissue

[0080] Malignant Lymphomas—Hodgkins Lymphoma

[0081] Non-Hodgkin's Lymphoma—Follicular lymphoma

[0082] Diffuse lymphoma

[0083] Small lymphocytic lymphoma

[0084] Large cell lymphoma

[0085] Lymphoblastic lymphoma

[0086] Multiple myeloma

[0087] Tumors of Connective Tissue

[0088] Cancers of Bone

[0089] Osteosarcoma

[0090] Tumors of the Nervous System

[0091] Neuroblastoma

[0092] Retinoblastoma

[0093] Glioblastoma

[0094] Oligodendroglioma

[0095] Tumors associated with oncogenic viruses

[0096] Human Papillomavirus—squamous cell carcinoma of cervix

[0097] Ebstein-Barr Virus—Burkitts Lymphoma

[0098] B cell lymphoma's in immuno-comprised individuals

[0099] Nasopharyngeal carcinoma

[0100] Hepatitis B Virus—Hepatocellular carcinoma

[0101] Herpes Virus 8 or Kaposi Sarcoma Herpes Virus (KSHV)—Kaposi'sSarcoma, and the like. Other neoplastic diseases known to the skilledartisan are also encompassed by the present invention including cancerof the oral cavity, larynx, kidney, testis and ovary. The skilledartisan will recognize that other cancers may be treated in accordancewith the present invention.

[0102] The term “a method for treating primary and metastatic tumors ofthe central nervous system” as used herein means that the disease andthe symptoms associated with the disease are alleviated, reduced, cured,or otherwise placed in a state of remission.

[0103] As used herein, the terms “a method for treating primary ormetastatic breast, lung, bladder or prostate cancer” and “a method fortreating metastases from breast, lung, bladder or prostate cancer” meansthat the disease and the symptoms associated with the disease arealleviated, reduced, cured, or placed in a state of remission. Inaddition, the term “a method for treating metastases from breast, lung,bladder or prostate cancer” means that the metastatic tumors and thesymptoms associated with the disease are alleviated, reduced, cured,placed in a state of remission.

[0104] The term “refractory” when used herein means that malignanciesare generally resistant to treatment or cure. The term “refractory” whenused in the above terms, means that the malignancies which are generallyresistant to treatment or cure are alleviated, reduced, cured, or placedin a state of remission.

[0105] As used herein the terms “a therapeutic agent”, “therapeuticregimen”, “radioprotectant”, “chemotherapeutic” mean conventional drugsand drug therapies, including vaccines, for treating cancer, viralinfections, and other malignancies, which are known to those skilled inthe art. “Radiotherapeutic” agents are well known in the art.

[0106] As used herein, “a method of treating cancer” or “a method oftreating solid tumors” or “a method of treating neoplastic diseases”means that the disease and the symptoms associated with the disease arealleviated, reduced, cured, or placed in a state of remission. Further,tumor growth is inhibited and/or tumor size is reduced.

[0107] As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions (See. Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B.Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form ofcontrolled cell proliferation involving an increase in cell number in atissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder. Although preneoplastic lesions may progress to neoplasia, theymay also remain stable for long periods and may even regress,particularly if the inciting agent is removed or if the lesion succumbsto an immunological attack by its host.

[0108] The therapeutic regimens and pharmaceutical compositions of theinvention may be used with additional immune response enhancers orbiological response modifiers including, but not limited to, thecytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, or otherimmunostimulatants/immunomodulators. In accordance with this aspect ofthe invention, the arsenic compounds are administered in combinationtherapy with one or more of these agents.

5.1. Formulation

[0109] The arsenic compounds of the invention may be formulated intopharmaceutical preparations for administration to mammals for treatmentof cancer. Compositions comprising a compound of the inventionformulated in a compatible pharmaceutical carrier may be prepared,packaged, labelled for treatment of and used for the treatment of theindicated tumor, such as human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia(myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia); chronic leukemia and chronic lymphocytic leukemia; andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease. Alternatively, it can be labeled for treatment of theappropriate infectious disease. Alternatively, pharmaceuticalcompositions may be formulated for treatment of appropriate infectiousdiseases.

[0110] If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions. Alternatively, if the resultingcomplex has poor solubility in aqueous solvents, then it may beformulated with a non-ionic surfactant such as Tween, polyethyleneglycol or glycerine. Thus, the compounds and their physiologicallyacceptable solvates may be formulated for administration by inhalationor insufflation (either through the mouth or the nose) or oral, buccal,parenteral, topical, dermal, vaginal, drug delivery device, e.g., porousor viscous material such as lipofoam, rectal administration or, in thecase of tumors, directly injected into a solid tumor.

[0111] For oral administration, the pharmaceutical preparation may be inliquid form, for example, solutions, syrups or suspensions, or may bepresented as a drug product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

[0112] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0113] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0114] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0115] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Suchformulations are sterile. Formulations for injection may be presented inunit dosage form, e.g., in ampules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0116] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0117] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Liposomes and emulsions are wellknown examples of delivery vehicles or carriers for hydrophilic drugs.

[0118] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0119] The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the arsenic compounds inpharmaceutically acceptable form. The arsenic compound in a vial of akit of the invention may be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the complex may be lyophilized or desiccated; inthis instance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

[0120] In another embodiment, a kit of the invention further comprises aneedle or syringe, preferably packaged in sterile form, for injectingthe complex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of arsenic compounds by a clinician or bythe patient.

[0121] The magnitude of a therapeutic dose of an arsenic compound in theacute or chronic management of cancer will vary with the severity of thecondition to be treated and the route of administration. The dose, andperhaps dose frequency, will also vary according to the age, bodyweight, condition and response of the individual patient. In general,the total daily dose ranges for the conditions described herein aregenerally from about 10 μg to about 200 mg administered in divided dosesadministered parenterally or orally or topically. A preferred totaldaily dose is from about 0.5 mg to about 70 mg of the active ingredient.

[0122] Desirable blood levels may be maintained by a continuous infusionof an arsenic compound as ascertained by plasma levels. It should benoted that the attending physician would know how to and when toterminate, interrupt or adjust therapy to lower dosage due to toxicity,or bone marrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

[0123] Again, any suitable route of administration may be employed forproviding the patient with an effective dosage of an arsenic compound.For example, oral, rectal, vaginal, transdermal, parenteral(subcutaneous, intramuscular, intrathecal and the like) may be employed.Dosage forms include tablets, troches, cachet, dispersions, suspensions,solutions, capsules, patches, and the like. (See, Remington'sPharmaceutical Sciences.)

[0124] The pharmaceutical compositions of the present invention comprisean arsenic compound as the active ingredient, or a pharmaceuticallyacceptable salt thereof, and may also contain a pharmaceuticallyacceptable carrier, and optionally, other therapeutic ingredients, forexample antivirals. The term “pharmaceutically acceptable salts” refersto salts prepared from pharmaceutically acceptable non-toxic acids andbases, including inorganic and organic acids and bases.

[0125] The pharmaceutical compositions include compositions suitable fororal, rectal, mucosal routes, transdermal, parenteral (includingsubcutaneous, intramuscular, intrathecal and intravenous), although themost suitable route in any given case will depend on the nature andseverity of the condition being treated.

[0126] In the case where an intravenous injection or infusioncomposition is employed, a suitable dosage range for use is, e.g., fromabout 0.5 mg to about 150 mg total daily dose.

[0127] In addition, the arsenic carrier could be delivered via chargedand uncharged matrices used as drug delivery devices such as celluloseacetate membranes, also through targeted delivery systems such-asfusogenic liposomes attached to antibodies or specific antigens.

[0128] In practical use, an arsenic compound can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including tablets, capsules, powders, intravenous injections orinfusions). In preparing the compositions for oral dosage form any ofthe usual pharmaceutical media may be employed, e.g., water, glycols,oils, alcohols, flavoring agents, preservatives, coloring agents, andthe like; in the case of oral liquid preparations, e.g., suspensions,solutions, elixirs, liposomes and aerosols; starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like in the case of oral solidpreparations e.g., powders, capsules, and tablets. In preparing thecompositions for parenteral dosage form, such as intravenous injectionor infusion, similar pharmaceutical media may be employed, e.g., water,glycols, oils, buffers, sugar, preservatives and the like know to thoseskilled in the art. Examples of such parenteral compositions include,but are not limited to Dextrose 5% w/v, normal saline or othersolutions. The total dose of the arsenic compound may be administered ina vial of intravenous fluid, e.g., ranging from about 2 ml to about 2000ml. The volume of dilution fluid will vary according the total doseadministered.

5.2. Target Cancers

[0129] Cancers that can be treated by the methods of the presentinvention include, but not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia(myeloblastic, myelomonocytic, monocytic and erythroleukemia); andchronic lymphocytic leukemia; and polycythemia vera, lymphoma (Hodgkin'sdisease and nonHodgkin's disease), multiple myeloma, Waldenström'smacroglobulinemia, and heavy chain disease. Specific examples of suchcancers are described in the sections below.

[0130] In a specific embodiment the cancer is metastatic. In anotherspecific embodiment, the patient having a cancer is immunosuppressed byreason of having undergone anti-cancer therapy (e.g., chemotherapyradiation) prior to administration of the arsenic compounds of theinvention.

[0131] In a specific embodiment, the present invention providescompositions and methods for enhancing tumor specific immunity inindividuals suffering from colorectal cancer metastasized to the liver,in order to inhibit the progression of the neoplastic disease. Preferredmethods of treating these neoplastic diseases comprise administering acomposition of arsenic which elicits an immune response against tumorcells.

[0132] In another specific embodiment, the present invention providescompositions and methods for enhancing specific immunity in individualssuffering from hepatocellular carcinoma in order to inhibit theprogression of the neoplastic disease and ultimately irradiate allpreneoplastic and neoplastic cells.

[0133] In a specific embodiment, the present invention provides hspcompositions and methods for enhancing specific immunity topreneoplastic and neoplastic mammary cells in women. The presentinvention also provides compositions and methods for inhibiting cancercell proliferation and metastasis. These compositions can be appliedalone or in combination with each other or with biological responsemodifiers.

6. WORKING EXAMPLES

[0134] The following subsections describe the testing of apharmaceutical composition comprising arsenic trioxide in vitro using apanel of cancer cell lines employed by the National Cancer Institute(NCI). The results demonstrate that arsenic trioxide is effective ininhibiting the growth of a broad range of leukemic cells and cancercells in vitro.

6.1. Methods and Materials

[0135] Arsenic trioxide (1 mg/ml, 10 mg/ampoule, manufactured by TaylorPharmaceuticals, Decatur, Ill.) was tested at five concentrations eachat 10-fold dilutions, i.e., 10^(−5, 10) ^(−6, 10) ⁻⁷, 10⁻⁸, and 10⁻⁹μg/ml.

[0136] The in vitro tests were performed by incubating the test cells inthe presence of the indicated concentration of arsenic trioxide understandard culture conditions for a designated period of time, which isfollowed by a sulforhodamine B (SRB) protein assay to estimate cellviability or growth. The cell lines are organized into subpanelsaccording to the origin of the cell lines, e.g., leukemia, breastcancer, etc. A description of the cell lines and method of testing isdescribed in Monk et al. (1997, Anticancer Drug Des. 12:533-41) andWeinstein et al., (1997, Science 275:343-9), which are incorporatedherein in their entirety.

[0137] Described below are the data analysis procedures and displays.

[0138] The measurement of an effect is expressed in Percentage Growth(PG). The measured effect of the compound on a cell line is calculatedaccording to one or the other of the following two expressions:

If (Mean OD _(test)−Mean OD _(zero))≧0. then PG=100×(Mean OD_(test)−Mean OD _(zero))/(Mean OD _(ctrl)−Mean OD _(zero))

If (Mean OD _(test)−Mean OD _(zero))<0. then PG=100×(Mean OD_(test)−Mean OD _(zero))/Mean OD _(zero)

[0139] where:

[0140] Mean OD_(zero)=the average of optical density measurements ofSRB-derived color just before exposure of cells to the test compounds;

[0141] Mean OD_(test)=the average of optical density measurements ofSRB-derived color after exposure of cells to the test compound for adesignated period of time; and

[0142] Mean OD_(ctrl)=the average of optical density measurements ofSRB-derived color after with no exposure of cells to the test compoundfor a designated period of time

[0143] Table 1 and 2 present the experimental data collected againsteach cell line. The first two columns describe the subpanel (e.g.,leukemia) and cell line (e.g., CCRF-CEM) involved. The next two columnslist the Mean OD_(zero) and Mean OD_(ctrl); the next five columns listthe Mean OD_(test) for each of five different concentrations. Eachconcentration is expressed as the log₁₀ (molar or μg/ml). The next fivecolumns list the calculated PGs for each concentration. The responseparameters GI50, TGI and LC50 are interpolated values representing theconcentrations at which the PG is +50, 0, and −50, respectively.Sometimes these response parameters cannot be obtained by interpolation.If, for instance, all of the PGs in a given row exceed +50, then none ofthe three parameters can be obtained by interpolation. In such a case,the value given for each response parameter is the highest concentrationtested and is preceded by a “>” sign. This practice is extendedsimilarly to the other possible situations where a response parametercannot be obtained by interpolation.

[0144] A dose-response curve (see FIGS. 1A-1I and 3A-3I) for the set ofdata is created by plotting the PGs against the log₁₀ of thecorresponding concentration for every cell line. The cell line curvesare grouped by subpanel. Horizontal lines are provided at the PG valueof +50, 0, and −50. The concentrations corresponding to points where thecurves cross these lines are the G150, TGI, and LC50, respectively.

[0145] A mean graph (FIGS. 2 and 4) facilitates visual scanning of datafor potential patterns of selectively for particular cell lines or forparticular subpanels with respect to a selected response parameter.Differences in apparent selectivity patterns may occur for the samecompound against the same cell lines when different parameters arecompared. The mean graphs page of the data package shows mean graphs ateach of the principal response parameters: GI50, TGI, and LC50. Barsextending to the right represent sensitivity of the cell line to thetest agent in excess of the average sensitivity of all tested celllines. Since the bar scale is logarithmic, a bar 2 units to the rightimplies the compound achieved the response parameter (e.g., GI50) forthe cell line at a concentration one-hundredth the mean concentrationrequired over all cell lines, and thus the cell line is unusuallysensitive to that compound. Bars extending to the left correspondinglyimply sensitivity less than the mean. If for a particular drug and cellline, it was not possible to determine the desired response parameter byinterpolation, the bar length shown is either the highest concentrationtested (and the listed log₁₀ of the response parameter will be precededby a “>”) or the lowest concentration tested (and the listed log₁₀ willbe preceded by a “<”).

[0146] The values at either limit (>or <) are also calculated in themean used for the mean graph. Therefore, the mean used in the mean graphmay not be the actual mean of the GI50 for instance. For this reason,this value is referred to as the MG_MID (for mean graph midpoint).

6.2. Results

[0147] The results of two sets of tests is presented below. In the firstset, the cells from 56 different cancer cell lines were exposed to fiveconcentrations of arsenic trioxide continuously for two days prior toperforming the SRB assay. In the second set, the cells from 50 differentcell lines (a subset of the first 56 cell lines, plus the renal cancercell line 786-0) were exposed continuously for six days prior to the SRBassay. TABLE 1 Log 10 Concentration Time Mean Optical Densities PercentGrowth Zero Ctrl −8.9 −7.9 −6.9 −5.9 −4.9 −8.9 −7.9 −6.9 −5.9 −4.9 GI50TGI LC50 Leukemia CCRF-CEM 0.300 1.155 1.203 1.195 1.134 0.704 0.285 106105 98 47 −5 1.11E−06  1.00E−05 >1.26E−05 HL-60(TB) 0.233 0.530 0.5330.507 0.535 0.499 0.213 101 92 102 90 −9 3.19E−06  1.03E−05 >1.26E−05K-562 0.209 1.416 1.387 1.431 1.418 1.124 0.199 98 101 100 76 −52.63E−06  1.10E−05 >1.26E−05 MOLT-4 0.134 0.438 0.465 0.454 0.454 0.3680.146 109 105 105 77 4 2.96E−06 >1.26E−05 >1.26E−05 RPMI-8226 0.2570.893 0.868 0.848 0.813 0.670 0.204 96 93 87 65 −21 1.88E−06 7.23E−06 >1.26E−05 SR 0.158 0.457 0.454 0.425 0.457 0.338 0.111 99 89100 60 −30 1.63E−06  5.85E−06 >1.26E−05 Non-Small Cell Lung CancerA549/ATCC 0.015 0.477 0.479 0.486 0.476 0.516 0.336 100 102 100 10869 >1.26E−05  >1.26E−05 >1.26E−05 EKVX 0.342 0.736 0.809 0.849 0.8410.853 0.385 119 129 127 130 11 5.91E−06 >1.26E−05 >1.26E−05 HOP-62 0.3351.109 1.088 1.099 1.113 1.086 0.605 97 99 100 97 357.18E−06 >1.26E−05 >1.26E−05 HOP-92 0.505 1.694 1.554 1.603 1.477 1.3810.873 88 92 82 74 31 4.50E−06 >1.26E−05 >1.26E−05 NCI-H226 0.560 0.9320.967 0.918 0.967 0.967 0.904 109 96 109 10992 >1.26E−05  >1.26E−05 >1.26E−05 NCI-H23 0.648 1.622 1.769 1.822 1.8801.635 1.215 115 121 127 101 58 >1.26E−05  >1.26E−05 >1.26E−05 NCI-8322M0.382 0.997 1.103 1.036 0.976 0.992 0.755 117 106 97 9961 >1.26E−05  >1.26E−05 >1.26E−05 NCI-H460 0.296 1.235 1.132 1.186 1.2341.157 0.757 89 95 100 92 49 1.20E−05 >1.26E−05 >1.26E−05 NCI-H522 0.4781.138 1.332 1.135 1.189 0.892 0.378 129 100 108 63 −21 1.79E−06 7.08E−06 >1.26E−05 Colon Cancer COLO 205 0.328 1.394 1.425 1.414 1.5761.434 0.935 103 102 117 104 57 >1.26E−05  >1.26E−05 >1.26E−05 HCT-1160.301 1.574 1.508 1.480 1.488 1.391 0.685 95 93 93 86 305.53E−06 >1.26E−05 >1.26E−05 HCT-15 0.219 1.623 1.800 1.627 1.673 1.5220.504 113 100 104 93 20 4.90E−06 >1.26E−05 >1.26E−05 HT29 0.095 0.5780.637 0.599 0.580 0.479 0.169 112 104 100 80 153.63E−06 >1.26E−05 >1.26E−05 KM12 0.189 0.741 0.744 0.728 0.777 0.7370.567 101 98 106 99 68 >1.26E−05  >1.26E−05 >1.26E−05 SW-620 0.153 0.8860.898 0.868 0.857 0.779 0.267 102 98 96 85 164.05E−06 >1.26E−05 >1.26E−05 CNS Cancer SF-268 0.203 0.767 0.821 0.7360.815 0.767 0.334 110 94 108 100 23 5.63E−06 >1.26E−05 >1.26E−05 SF-2950.249 1.007 0.951 0.978 0.938 0.938 0.639 93 96 91 9151 >1.26E−05  >1.26E−05 >1.26E−05 SF-539 0.132 0.462 0.491 0.516 0.5060.435 0.110 109 117 113 92 −17 3.06E−06  8.85E−06 >1.26E−05 SNB-19 0.1760.905 0.857 0.880 0.887 0.883 0.437 93 97 98 97 367.38E−06 >1.26E−05 >1.26E−05 SNB-75 0.501 1.051 0.925 1.049 1.134 0.7030.438 77 100 115 37 −13 8.54E−07  6.98E−06 >1.26E−05 U251 0.192 0.7990.789 0.792 0.803 0.680 0.061 98 99 101 80 −68 2.02E−06  4.37E−06 9.47E−06 Melanoma LOX IMVI 0.173 1.304 1.175 1.170 0.974 0.997 0.319 8988 71 73 13 3.03E−06 >1.26E−05 >1.26E−05 MALME-3M 0.476 0.859 1.0060.949 0.868 0.819 0.275 138 123 102 90 −42 2.51E−06  6.02E−06 >1.26E−05M14 0.123 0.613 0.602 0.635 0.592 0.549 0.136 98 105 96 87 33.45E−06 >1.26E−05 >1.26E−05 SK-MEL-2 0.388 0.704 0.746 0.714 0.7350.676 0.323 113 103 110 91 −17 3.03E−06  8.81E−06 >1.26E−05 SK-MEL-280.513 1.090 1.088 1.107 1.093 1.057 0.546 100 103 101 94 63.98E−06 >1.26E−05 >1.26E−05 SK-MEL-5 0.454 2.107 2.049 2.066 2.0251.748 0.460 96 97 95 78 0 2.90E−06 >1.26E−05 >1.26E−05 UACC-257 0.5961.142 1.149 1.128 1.165 1.078 0.814 101 97 104 88 407.78E−06 >1.26E−05 >1.26E−05 UACC-62 0.306 1.082 1.111 1.105 1.108 1.0510.355 104 103 103 96 6 4.10E−06 >1.26E−05 >1.26E−05 Ovarian CancerIGROV1 0.291 1.679 1.828 1.932 1.743 1.710 0.536 111 118 105 102 185.22E−06 >1.26E−05 >1.26E−05 OVCAR-3 0.240 1.066 1.073 1.055 1.045 0.9800.343 101 99 97 90 12 4.10E−06 >1.26E−05 >1.26E−05 OVCAR-5 0.457 1.2061.243 1.230 1.157 1.181 0.715 105 103 93 97 347.07E−06 >1.26E−05 >1.26E−05 OVCAR-8 0.173 1.340 1.250 1.245 1.159 1.1070.495 92 92 84 80 28 4.71E−06 >1.26E−05 >1.26E−05 SK-OV-3 0.351 0.8650.836 0.852 0.853 0.867 0.557 94 97 98 100 408.59E−06 >1.26E−05 >1.26E−05 Renal Cancer A498 0.400 0.678 0.691 0.6570.649 0.635 0.357 105 93 89 84 −11 2.90E−06  9.71E−06 >1.26E−05 CAKI-10.372 0.942 1.058 0.960 1.103 0.819 0.325 120 103 128 78 −13 2.59E−06 9.13E−06 >1.26E−05 RXE 393 0.743 1.243 0.860 1.114 1.290 1.077 0.776 2374 109 67 7 . >1.26E−05 >1.26E−05 SN12C 0.157 0.546 0.533 0.529 0.5000.474 0.113 97 96 88 81 −28 2.44E−06  6.96E−06 >1.26E−05 TX-10 0.3551.027 1.014 1.002 1.042 0.999 0.727 98 96 102 9655 >1.26E−05  >1.26E−05 >1.26E−05 UO-31 0.124 0.765 0.799 0.816 0.8300.785 0.391 105 108 110 103 42 9.20E−06 >1.26E−05 >1.26E−05 ProstateCancer PC-3 0.187 0.419 0.426 0.399 0.390 0.342 0.179 103 91 88 67 −42.17E−06  1.10E−05 >1.26E−05 DU-145 0.384 1.151 1.081 1.389 1.194 1.1190.873 91 131 106 96 64 >1.26E−05  >1.26E−05 >1.26E−05 Breast Cancer MCF70.200 0.928 0.924 0.912 0.985 0.893 0.277 99 98 108 95 114.31E−06 >1.26E−05 >1.26E−05 NCI/ADR-RES 0.328 1.386 1.576 1.376 1.4251.298 0.801 118 99 104 92 45 9.69E−06 >1.26E−05 >1.26E−05 MDA-MB- 0.3040.644 0.635 0.651 0.674 0.667 0.314 97 102 109 107 34.43E−06 >1.26E−05 >1.26E−05 231/ATCC HS 578T 0.369 1.255 1.338 1.2961.219 1.300 0.742 109 105 96 105 42 9.44E−06 >1.26E−05 >1.26E−05MDA-MB-435 0.465 1.425 1.370 1.354 1.476 1.369 0.666 94 93 105 94 215.05E−06 >1.26E−05 >1.26E−05 MDA-N 0.348 1.471 1.499 1.481 1.374 1.3160.385 102 101 91 86 3 3.44E−06 >1.26E−05 >1.26E−05 BT-549 0.735 1.7621.910 1.865 1.847 1.599 0.998 114 110 108 84 264.82E−06 >1.26E−05 >1.26E−05 T-47D 0.464 1.007 0.941 1.054 1.184 0.9050.419 88 109 133 81 −10 2.78E−06  9.86E−06 >1.26E−05

[0148] In the first set of tests, according to Table 1 and the doseresponse curves as shown in FIGS. 1A to 1I, arsenic trioxide waseffective in reducing cell growth against almost all the cell linestested. In particular, leukemic cell lines, melanoma cell lines, andovarian cancer cell lines responded consistently by showing a reductionof more than 50% growth. According to the mean graph as shown in FIG. 2,most of the leukemic and melanoma cell lines, central nervous systemcancer cell lines SNB-75 and U251., prostate cancer cell line PC-3,renal cancer cell lines A498, CAKI-1, SN12C, and lung cancer cell lineNC1-H522 were especially sensitive (relative to all the cell linestested) to treatment with arsenic trioxide. TABLE 2 Log 10 ConcentrationTime Mean Optical Densities Percent Growth Panel/Cell Line Zero Ctrl−8.9 −7.9 −6.9 −5.9 −4.9 −8.9 −7.9 −6.9 −5.9 −4.9 GI50 TGI LC50 LeukemiaCCRF-CEM 0.047 3.511 3.601 3.592 3.051 0.320 0.048 103 102 87 8 03.68E−07 >1.26E−05  >1.261−05  K-562 0.041 3.011 3.042 2.992 3.360 2.9310.096 101 99 112 97 2 3.941−06 >1.26E−05  >1.26E−05  MOLT-4 0.018 0.5030.554 0.228 0.432 0.245 0.113 111 43 85 47 20 >1.26E−05  >1.26E−05 RPMI-8226 0.066 1.432 1.607 1.263 1.423 0.368 0.065 113 88 99 22 −25.481−07 1.091−05 >1.26E−05  Non-small Cell Lung Cancer EKVX 0.030 0.6820.573 0.601 0.628 0.414 0.014 83 88 92 59 −53 1.51E−06 4.22E−06 1.18E−05HOP-62 0.061 1.026 1.076 0.997 1.019 0.451 0.014 105 97 99 40 −778.66E−07 2.781−06 7.41E−06 HOP-92 0.115 1.292 1.455 1.834 1.319 1.2290.079 114 146 102 95 −31 2.85E−06 7.111−06 >1.26E−05  NCI-H226 0.0940.498 0.504 0.458 0.489 0.348 0.164 101 90 98 63 172.40E−08 >1.261−05  >1.26E−05  NCI-H23 0.075 1.624 1.818 1.814 1.8721.303 0.086 113 112 116 79 1 2.97E−06 >1.261−05  >1.26E−05  NCI-H322M0.035 0.702 0.680 0.620 0.674 0.552 0.054 97 88 96 77 32.94E−06 >1.261−05  >1.26E−05  NCI-H460 0.034 2.839 2.797 2.806 2.7922.535 0.062 99 99 98 89 1 3.50E−06 >1.261−05  >1.26E−05  NCI-H522 0.1751.224 1.269 0.535 1.191 0.128 0.028 104 34 97 −27 −84 7.641−07 3.19E−06Colon Cancer COLO 205 0.022 2.351 2.308 2.026 2.283 1.404 0.024 98 86 9759 0 1.81E−06 >1.26E−05  >1.26E−05  HCT-116 0.046 3.420 3.395 3.2893.381 3.284 1.931 99 96 99 96 56 >1.26E−05  >1.26E−05  >1.26E−05  HCT-150.033 3.838 3.965 3.921 4.227 3.432 0.404 103 102 110 89 103.93E−06 >1.26E−05  >1.26E−05  HT29 0.017 3.060 3.189 2.988 2.706 1.4760.027 104 98 88 48 0 1.12E−06 >1.26E−05  >1.26E−05  KM12 0.012 0.8080.487 0.533 0.507 0.036 0.017 80 87 83 4 13.29E−07 >1.26E−05  >1.26E−05  SW-620 0.023 2.652 2.613 2.308 2.3371.475 0.007 99 87 88 55 −72 1.39E−06 3.43E−06 8.50E−06 CNS Cancer SF-2680.054 0.798 0.990 0.381 0.356 0.387 −0.004 126 44 41 45 −100 1.06E−082.57E−06 5.69E−06 SF-295 0.031 1.324 1.284 1.279 1.244 0.659 0.010 97 9794 49 −69 1.17E−06 3.25E−06 8.64E−06 SF-539 0.041 1.598 1.727 1.4031.545 0.691 0.017 108 87 97 42 −59 8.91E−07 3.29E−06 1.04E−05 SNB-750.134 1.065 1.143 1.033 1.024 0.815 0.028 108 97 96 73 −79 1.79E−063.80E−06 8.08E−06 U251 0.105 2.324 2.129 2.018 1.908 0.088 −0.012 91 8681 −17 −100 2.63E−07 8.51E−07 3.16E−06 Melanoma LOX IMVI 0.013 2.1532.000 1.666 0.053 0.002 −0.001 93 77 2 −88 −100 2.90E−08 1.32E−074.73E−07 MALMI-3M 0.115 0.650 0.702 0.595 0.823 0.383 0.005 110 90 13250 −96 1.26E−06 2.78E−06 6.12E−06 SK-MEL-2 0.213 0.500 0.491 0.462 0.4260.128 . 97 87 74 −40 −100 2.06E−07 5.63E−07 1.85E−06 SK-MEL-28 0.0932.030 2.012 1.906 1.797 0.488 . 99 94 88 20 −100 4.60E−07 1.86E−064.84E−06 SK-MEL-5 0.181 1.907 1.935 1.864 1.929 1.846 0.013 102 97 10196 −93 2.22E−06 4.07E−06 7.46E−06 UACC-257 0.053 0.883 0.872 0.857 0.8320.504 0.039 99 97 94 54 −27 1.42E−06 5.83E−06 >1.26E−05  UACC-62 0.0301.537 1.546 1.460 1.409 0.539 0.001 101 95 92 34 −98 6.60E−07 2.27E−065.43E−06 Ovarian Cancer IGROV1 0.335 3.115 3.541 3.056 3.204 2.877 0.216115 98 103 91 −36 2.67E−06 6.62E−06 >1.26E−05  OVCAR-3 0.081 1.153 1.0471.128 1.124 0.482 0.001 90 98 97 37 −99 7.76E−07 2.36E−06 5.49E−06OVCAR-5 0.038 1.404 1.433 1.460 1.478 1.287 0.028 102 104 105 91 −262.83E−06 7.53E−06 >1.26E−05  OVCAR-8 0.016 2.090 2.272 1.848 2.242 1.437−0.001 109 88 107 68 −100 1.62E−06 3.21E−06 6.36E−06 SK-OV-3 0.041 1.4741.445 1.410 1.461 1.247 0.020 98 96 99 84 −51 2.25E−06 5.27E−06 1.23E−05Renal Cancer 786-0 0.021 1.578 1.582 1.821 1.786 0.832 −0.003 100 116113 52 −100 1.30E−06 2.77E−06 5.91E−06 A498 0.051 0.741 0.826 0.7640.837 0.558 0.025 112 103 114 73 −52 1.94E−06 4.85E−06 1.22E−05 CAKI-10.032 1.352 1.254 1.725 1.573 0.491 0.004 93 128 117 35 −88 8.22E−072.43E−06 6.22E−06 RXF 393 0.198 1.515 1.327 1.301 1.727 0.909 0.017 8684 116 54 −91 1.34E−06 2.96E−06 6.54E−06 S12C 0.024 0.956 0.882 1.1411.113 0.829 0.013 92 120 117 86 −46 2.37E−06 5.67E−06 >1.26E−05  TK-100.072 1.333 1.356 1.268 1.334 1.103 0.239 102 95 100 82 133.66E−06 >1.26E−05  >1.26E−05  Prostate Cancer DU-145 0.095 2.158 2.1471.954 1.935 2.153 0.055 99 90 89 100 −43 2.82E−06 6.32E−06 >1.26E−05 Breast Cancer MCF7 0.028 2.650 2.647 2.611 2.603 1.683 0.008 100 99 9863 −73 1.57E−06 3.66E−06 8.51E−06 NCI/ADR-RIS 0.050 2.304 2.075 2.2531.951 1.319 0.072 90 98 84 56 1 1.64E−06 >1.26E−05  >1.26E−05  MDA-MB-0.158 0.761 0.759 0.764 0.771 0.563 −0.001 100 101 102 67 −100 1.59E−063.18E−06 6.33E−06 231/ATCC HS 578T 0.109 0.728 0.777 0.587 0.890 0.7060.010 108 77 126 96 −91 2.23E−06 4.12E−06 7.62E−06 MDA-MB-435 0.0541.500 1.276 1.470 0.865 0.631 0.021 85 98 56 40 −62 3.00E−07 3.10E−069.60E−06 MDA-N 0.028 2.652 2.468 2.616 2.606 1.738 0.026 93 99 98 65 −92.02E−06 9.55E−06 >1.26E−05  BT-549 0.112 1.798 1.876 1.940 1.963 1.6970.018 105 108 110 94 −84 2.22E−06 4.24E−06 8.08E−06 T-47D 0.098 0.8240.883 0.336 0.539 0.398 0.027 108 33 61 41 −73 . 2.90E−06 7.93E−06

[0149] In the second set of tests, according to Table 2, the doseresponse curves, and the mean graph as shown in FIGS. 3A to 3I, and FIG.4, arsenic trioxide was effective in reducing cell growth against allthe cell lines tested. The results were consistent with those obtainedfrom the first set of tests. In particular, several melanoma cell linesappeared to be especially sensitive at the various principal responseparameters.

[0150] In conclusion, these results demonstrate that arsenic trioxide iseffective in inhibiting the growth of leukemic cells and cancer cells invitro, and that arsenic trioxide can be used in human subjects to treata broad range of leukemia, and cancers, including but not limited tonon-small cell lung cancer, colon cancer, central nervous system cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, and breastcancer.

[0151] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method of treating lymphoma in a human, which comprisesadministering a therapeutically effective amount of one or more arseniccompounds to said human.
 2. The method of claim 1, wherein said arseniccompound is arsenic trioxide.
 3. The method of claim 2, wherein saidarsenic trioxide is formulated as an ionic aqueous solution.
 4. Themethod of claim 1, wherein the total daily amount administered is fromabout 10 μg to about 200 mg.
 5. The method of claim 1, wherein the totaldaily amount administered is from about 0.5 mg to about 150 mg.
 6. Themethod of claim 1, wherein the total daily amount administered is fromabout 0.5 mg to about 70 mg.
 7. The method of claim 1, wherein thearsenic compound is administered parenterally.
 8. The method of claim 1,wherein the arsenic compound is administered intravenously.
 9. Themethod of claim 1, wherein the arsenic compound is administered incombination with an effective amount of at least one other therapeutic.10. The method of claim 9, wherein the other therapeutic agent is achemotherapeutic or radiotherapeutic.
 11. The method of claim 9, whereinthe other therapeutic agent is selected from the group consisting ofetoposide, cisplatin, carboplatin, estramustine phosphate, vinblastine,methotrexate, hydroxyurea, cyclophosphamide, doxorubicin,5-fluorouracil, taxol, diethylstilbestrol, VM-26(vumon), BCNU, all-transretinoic acid, procarbazine, cytokines, therapeutic vaccines, andimmunomodulators.
 12. The method of claim 2, wherein the dose is variedaccording to the body weight of said human.
 13. The method of claim 1,wherein the lymphoma is Hodgkins lymphoma.
 14. The method of claim 1,wherein the lymphoma is non-Hodgkin's lymphoma.
 15. The method of claim1, wherein the lymphoma is follicular lymphoma.
 16. The method of claim1, wherein the lymphoma is diffuse lymphoma.
 17. The method of claim 1,wherein the lymphoma is lymphoblastic lymphoma.
 18. The method of claim1, wherein the lymphoma is large cell lymphoma.
 19. The method of claim1, wherein the lymphoma is small lymphocytic lymphoma.
 20. The method ofclaim 1, wherein the lymphoma is HIV-related nonHodgkin's lymphoma.